While head impact biomechanics studies have advanced our understanding of concussion, no studies to date have involved professional football players. PURPOSE: To examine differences in head impact biomechanics between 3 cohorts of offensive and defensive linemen: 1) National Football League (NFL), 2) College, and 3) High School (HS). METHODS: Players from 2 NFL teams (n=14; 6,127 total impacts), 1 college team (n=12; 7,138 total impacts), and 1 HS team (n=10; 4,136 total impacts) were enrolled in this study. The Head Impact Telemetry System (Riddell, Rosemont, IL) measured the following head impact variables: linear acceleration, rotational acceleration, and impact location. Data underwent a natural logarithmic transformation to normalize acceleration data prior to our analyses. We applied general linear mixed models to our acceleration variables and Chi Square tests to our head impact locations. RESULTS: We observed no significant differences for linear acceleration (F2,33=2.38, p=0.109) or rotational acceleration (F2,33=1.67, p=0.205) between cohorts. Analyses of the 95th percentile for linear acceleration and rotational acceleration revealed similar values for linear acceleration (NFL=62.2g, college=62.6g, HS=63.4g) and rotational acceleration (NFL=3859.9 rad/s2, college=3824.0 rad/s2, HS=4092.4 rad/s2) between cohorts. We observed an association between impact locations and the three cohorts (χ2(6)=676.58, p<0.001). The HS linemen experienced a significantly higher frequency of impacts to the front of the head (NFL=37.7%, college=34.3%, HS=53.4%) and a significantly lower frequency of impacts to the top of the head (NFL=19.4%, college=26.0%, HS=9.2%). CONCLUSION: Our head impact acceleration findings were expected given the literature has not identified any differences between HS and college cohorts to date. Future studies should explore speed and skill positions across competitive level. Many behavioral interventions are designed to educate HS football players on safe tackling techniques and the importance of ‘seeing what they hit.’ Our data show HS linemen experience more front-of-head and fewer top-of-head impacts. Future studies should measure the efficacy of these programs in light of the encouraging HS data we observed. Supported by the National Football League.